EP2644699B1 - Verfahren und vorrichtung zum nachweis amplifizierter nukleinsäuren - Google Patents
Verfahren und vorrichtung zum nachweis amplifizierter nukleinsäuren Download PDFInfo
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- EP2644699B1 EP2644699B1 EP11843368.9A EP11843368A EP2644699B1 EP 2644699 B1 EP2644699 B1 EP 2644699B1 EP 11843368 A EP11843368 A EP 11843368A EP 2644699 B1 EP2644699 B1 EP 2644699B1
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- nucleic acid
- primer
- oligonucleotide probe
- dna fragment
- dna
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Claims (14)
- Nukleinsäure-Nachweisverfahren, umfassend den Schritt des Hybridisierens einer ersten Oligonukleotid-Sonde, die auf einer festen Phase immobilisiert ist, mit einem jener einzelsträngigen Bereiche, die an jeweils gegenüberliegenden Enden eines amplifizierten doppelsträngigen DNA-Fragments liegen und natürliche Nukleotide umfassen, und
den Schritt des Hybridisierens einer zweiten Oligonukleotid-Sonde, die mit einer Markierungssubstanz markiert ist, mit dem anderen einzelsträngigen Bereich des amplifizierten DNA-Fragments,
wobei das amplifizierte DNA-Fragment ein Produkt ist, das mit einem Nukleinsäure-Amplifikationsverfahren erhalten wurde, und zwar unter Verwendung von zwei Primern, von denen jeder einen Markierungsbereich, der durch eine Nukleinsäure-Amplifikationsreaktion nicht doppelsträngig gemacht wird, und ein Nukleotid enthält, das durch die folgende Formel (1) dargestellt ist - Nukleinsäure-Nachweisverfahren nach Anspruch 1, wobei die Markierungssubstanz einen gefärbten Träger umfasst, der es erlaubt, das amplifizierte DNA-Fragment visuell nachzuweisen.
- Nukleinsäure-Nachweisverfahren nach einem der Ansprüche 1 bis 2, umfassend den Schritt des Nachweisens des Vorhandenseins des amplifizierten DNA-Fragments auf einer Nukleinsäure-Nachweisvorrichtung.
- Nukleinsäure-Nachweisverfahren nach einem der Ansprüche 1 bis 3, wobei das Vorhandensein des amplifizierten DNA-Fragments durch Chromatographie nachgewiesen wird.
- Nukleinsäure-Nachweisverfahren nach Anspruch 3 oder 4, umfassend die folgenden Schritte (a) bis (c):(a) Platzieren des amplifizierten DNA-Fragments in einer Zone auf der Nukleinsäure-Nachweisvorrichtung, die sich von einer Zone unterscheidet, wo die erste Oligonukleotid-Sonde immobilisiert ist;(b) Diffundieren des amplifizierten DNA-Fragments mit einem Lösungsmittel auf der Vorrichtung in Richtung der Zone, wo die erste Oligonukleotid-Sonde immobilisiert ist; und(c) Hybridisieren der ersten Oligonukleotid-Sonde mit dem amplifizierten DNA-Fragment in der Zone, wo die erste Oligonukleotid-Sonde immobilisiert ist.
- Nukleinsäure-Nachweisverfahren nach Anspruch 5, ferner umfassend den Schritt des Hybridisierens der zweiten Oligonukleotid-Sonde, die mit der Markierungssubstanz markiert ist, mit dem amplifizierten DNA-Fragment vor Schritt (c).
- Nukleinsäure-Nachweisverfahren nach Anspruch 3 oder 4, umfassend die folgenden Schritte (d) bis (h):(d) Platzieren des amplifizierten DNA-Fragments bzw. der zweiten Oligonukleotid-Sonde, die mit der Markierungssubstanz markiert ist, in jeweils diskreten Zonen auf der Nukleinsäure-Nachweisvorrichtung, die sich von der Zone unterscheiden, wo die erste Oligonukleotid-Sonde immobilisiert ist;(e) Diffundieren des amplifizierten DNA-Fragments mit einem Lösungsmittel in Richtung der Zone wo die zweite Oligonukleotid-Sonde, die mit der Markierungssubstanz markiert ist, platziert ist;(f) Hybridisieren der zweiten Oligonukleotid-Sonde, die mit der Markierungssubstanz markiert ist, mit dem amplifizierten DNA-Fragment in der Zone, wo die zweite Oligonukleotid-Sonde, die mit der Markierungssubstanz markiert ist, platziert ist;(g) Diffundieren eines in Schritt (f) erhaltenen Hybridisierungskomplexes auf einem Entwicklungsmedium in Richtung der Zone, wo die erste Oligonukleotid-Sonde platziert ist; und(h) Hybridisieren der ersten Oligonukleotid-Sonde mit dem Komplex in der Zone, wo die erste Oligonukleotid-Sonde immobilisiert ist.
- Nukleinsäure-Nachweisverfahren nach einem der Ansprüche 1 bis 7, wobei jeder der einzelsträngigen Bereiche, der natürliche Nukleotide umfasst, eine Sequenz in derselben Orientierung wie der doppelsträngige DNA-Bereich hat.
- Nukleinsäure-Nachweisverfahren nach einem der Ansprüche 1 bis 8, wobei das amplifizierte DNA-Fragment ein Produkt ist, das durch ein Nukleinsäure-Amplifikationsverfahren erhalten wurde, und zwar unter Verwendung eines ersten Primer-Sets, das Primer einschließt, von denen jeder eine Sequenz, die an eine Matrize der Zielnukleinsäure hybridisieren kann und eine gemeinsame Sequenz umfasst, die nicht an die Matrize hybridisieren kann, und eines zweiten Primer-Sets, das Primer einschließt, von denen jeder eine Sequenz, die an eine Sequenz hybridisieren kann, die komplementär zur korrespondierenden gemeinsamen Sequenz ist, und eine Markierungsregion umfasst, die nicht durch eine Nukleinsäure-Amplifikationsreaktion doppelsträngig gemacht wird.
- Nukleinsäure-Nachweisverfahren nach Anspruch 9, wobei die Markierungsregion die nicht durch eine Nukleinsäure-Amplifikationsreaktion doppelsträngig gemacht wird, natürliche Nukleotide umfasst, und eine Sequenz in der gleichen Orientierung wie die Sequenz hat, die an eine Sequenz hybridisieren kann, die komplementär zur korrespondierenden gemeinsamen Sequenz ist.
- Nukleinsäure-Nachweisverfahren nach einem der vorangegangenen Ansprüche, wobei das Verfahren vor jedem Hybridisierungsschritt einen Schritt des Herstellens des amplifizierten doppelsträngigen DNA-Fragments mit dem Nukleinsäure-Amplifikationsverfahren unter Verwendung der Primer umfasst.
- Verwendung einer Nachweisvorrichtung im Nukleinsäure-Nachweisverfahren nach einem der Ansprüche 1 bis 11, wobei die Vorrichtung umfasst:eine Zone, wo das amplifizierte DNA-Fragment platziert wird;einen chromatographischen Träger, der die erste Oligonukleotid-Sonde aufweist, die an das amplifizierte DNA-Fragment binden kann; unddie zweite Oligonukleotid-Sonde, die mit der Markierungssubstanz markiert ist.
- Amplifiziertes doppelsträngiges DNA-Fragment, das mit einem Nukleinsäure-Amplifikations-Verfahren hergestellt ist, das zwei Primer verwendet, von denen jeder einen Markierungsbereich, dessen Länge 5 Basen bis 60 Basen ist und der nicht durch eine Nukleinsäure-Amplifikationsreaktion doppelsträngig gemacht wird, und ein Nukleotid enthält, das durch die folgende Formel (1) dargestellt ist
- Amplifiziertes doppelsträngiges DNA-Fragment nach Anspruch 13, wobei der Markierungsbereich jedes Primers, der nicht durch eine Nukleinsäure-Amplifikationsreaktion doppelsträngig gemacht wird, natürliche Nukleotide umfasst, und
die gesamte Sequenz jedes Primers in der gleichen Orientierung ist.
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JP2016073312A (ja) | 2016-05-12 |
KR101919001B1 (ko) | 2018-11-16 |
EP3348640A1 (de) | 2018-07-18 |
JPWO2012070618A1 (ja) | 2014-05-19 |
JP6513892B1 (ja) | 2019-05-15 |
EP3348640B1 (de) | 2019-09-11 |
EP2644699A4 (de) | 2014-05-21 |
CN103228785B (zh) | 2016-09-07 |
JP2019080581A (ja) | 2019-05-30 |
CN106244695A (zh) | 2016-12-21 |
US9920356B2 (en) | 2018-03-20 |
CN103228785A (zh) | 2013-07-31 |
JP6063069B2 (ja) | 2017-01-18 |
US20180155771A1 (en) | 2018-06-07 |
EP2644699A1 (de) | 2013-10-02 |
US10829805B2 (en) | 2020-11-10 |
WO2012070618A1 (ja) | 2012-05-31 |
US20140065725A1 (en) | 2014-03-06 |
JP6783590B2 (ja) | 2020-11-11 |
JP5869715B2 (ja) | 2016-02-24 |
JP2016195614A (ja) | 2016-11-24 |
JP2013247968A (ja) | 2013-12-12 |
CN106244695B (zh) | 2019-12-13 |
KR20130118331A (ko) | 2013-10-29 |
JP2015156872A (ja) | 2015-09-03 |
SG190377A1 (en) | 2013-06-28 |
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